Comparative Genomic and Transcriptomic Analyses of Mycobacterium kansasii Subtypes Provide New Insights Into Their Pathogenicity and Taxonomy

Abstract

Mycobacterium kansasii is an important opportunistic pathogen of humans and has a close phylogenetic relationship with Mycobacterium tuberculosis. Seven subtypes (I-VII) have been identified using molecular biology approaches, of which subtype I is the most frequent causative agent of human disease. To investigate the genotypes and pathogenic components of M. kansasii, we sequenced and compared the complete base-perfect genomes of different M. kansasii subtypes. Our findings support the proposition that M. kansasii "subtypes" I-VI, whose assemblies are currently available, should be considered as different species. Furthermore, we identified the exclusive presence of the espACD operon in M. kansasii subtype I, and we confirmed its role in the pathogenicity of M. kansasii in a cell infection model. The espACD operon is exclusively present in mycobacterial species that induce phagosomal rupture in host phagocytes and is known to be a major determinant of ESX1-mediated virulence in pathogenic mycobacteria. Comparative transcriptome analysis of the M. kansasii I-V strains identified genes potentially associated with virulence. Using a comparative genomics approach, we designed primers for PCR genotyping of M. kansasii subtypes I-V and tested their efficacy using clinically relevant strains of M. kansasii.

Keywords: M. kansasii subtypes; comparative genomics; espACD operon; non-tuberculous mycobacteria; virulence factor.

Figure 1

Circular map representation of the M. kansasii subtype II–subtype V genome showing BlastN similarities to M. kansasii subtype I. Each ring of the circle corresponds to a specific complete genome as referred to in the figure keys on the right. The espACD operon position in M. kansasii KAUST I is indicated by an arrow.

Figure 2

Comparative genomics study of the chromosomes of M. kansasii subtypes I to V. Venn Diagram of the ortholog groups and paralogue groups shared by (A) the five M. kansasii subtypes and (B) the five subtypes with M. tuberculosis H37Rv. The number represents the ortholog groups or paralogue groups present in the particular types.

Figure 3

Genotyping of M. kansasii using ITS and SNPs from the complete genome assemblies. The maximum-likelihood tree based on the internal transcribed spacer (ITS) sequences of 45 M. kansasii strains is shown on the right. The maximum-likelihood phylogenetic tree based on 135,969 SNPs from the core genome of M. kansasii subtypes is shown on the left. Bootstrap support values are indicated on the branch as a percentage of 100,000 replicates. The branch length is ignored for illustration purposes and displayed on each branch. The color code for each genotype: red: M. kansasii subtype I; purple: M. kansasii subtype II; yellow: M. kansasii subtype III; green: M. kansasii subtype IV; blue: M. kansasii subtype V; brown: M. kansasii subtype VI. The strains within M. kansasii subtype I are separated with black rectangles in the figure. The circles indicate the difference between the ITS and SNP genotyping method, and colors represent the genotypes suggested by ANI clustering.

Figure 4

(A) Gene expression profiles (log₂ fold change) of the differentially expressed genes of M. kansasii subtypes II-V in comparison to M. kansasii subtype I. The red color represents upregulated genes, and the green color represents downregulated genes compared with the control strain. padj < 0.01 and Log₂FC > 2 were used as the cutoff for the selection of the genes. (B) Pie-chart showing a subdivision of all differentially expressed genes based on functional categories.

Figure 5

Complementation of the espACD operon in M. kansasii subtype II with that from M. kansasii subtype I is crucial for its virulence. The functional complementation experiment revealed that the espACD operon plays a significant role in M. kansasii subtype II pathogenicity, at least in THP-1 cells. Intracellular CFU count showing the bacterial load in THP-1 macrophages infected with KAUST-I (•), KAUST-II (•), KAUST-II-pSMT3 (▴) and KAUST-II-pSMT3-espACD (▾) at a MOI of 5. The infected macrophages were lysed at 0, 24, 48, and 72 h time-points post-infection and three dilutions of the released mycobacterial cells were plated on 7H10 agar plates. CFU were counted and recorded after 15 days of plating. Experiments were performed with three replicates, and Student's t-test for significance was calculated with the level of significance shown (***highly significant difference, p < 0.01).

Figure 6

Agarose gel (2%) electrophoresis of diagnostic PCR tests for M. kansasii subtypes I-V. Lane M: DNA markers. The gDNA used for testing is labeled on the top, and the size of the ladder is labeled on the left side of each gel picture. The genotyping results for M. kansasii subtypes I-V are listed in (A–E). The clinical samples tested with the cocktail of primers DP1-5 is shown in (F). The 16S primer control is shown in the red box. NTC, Non-template control.